The present invention relates to nucleic acid constructs, cells transformed therewith and methods of utilizing such constructs and transformed cells for inducing hepatocytes proliferation and liver regeneration.
Liver Cirrhosis
Cirrhosis, is a disease of the liver, which results from injury to liver tissue. Cirrhosis is characterized by scar tissue formation throughout the organ; groups of cells termed regenerative nodules, surrounded by sheets of scar tissue, replace the normal tissue of the liver. The diseased organ may be unable to perform vital functions such as manufacturing proteins and removing harmful substances from the blood. The affected liver tissue may block the flow of blood through the liver, causing high pressure in blood vessels, which serve the liver (portal hypertension). This blockage can lead to gastro-esophageal bleeding and ascites and in addition can contribute to the development of encephalopathy.
Liver injury occurs as a result of a number of acute and chronic clinical conditions, including drug-induced hepatotoxicity, viral infections, vascular injury, autoimmune disease, and blunt trauma. In addition, patients subject to inborn metabolic abnormalities may be at risk for developing liver cirrhosis.
Liver cirrhosis and its related clinical syndromes is a major cause of morbidity and mortality both in the developed and developing countries (Podolsky et al, 1988; Sherlock et al, 1997; Villanueva et al, 1996). According to the National Institute of Diabetes and Digestive and Kidney Diseases about 25,000 Americans die from cirrhosis each year.
Currently, liver damage resulting from cirrhosis cannot be reversed, although complications resulting from such liver damage can be treated by a variety of treatment regimens, which are targeted at preventing or alleviating such complications. For example, ascites and edema, are treatable by a low-sodium diet or use of diuretics, infections are treatable via antibiotics, while blood pressure medication (e.g., a beta-blocker) can be utilized to reduce the occurrence of gastro-intestinal bleeding in portal hypertension cases.
In extreme cases of liver dysfunction, liver transplant procedures are necessary. Although liver transplants can reestablish normal liver function such procedures are complex and as such only successful in a fraction of the cases. In addition, constant shortage of organs suitable for transplantation further limits application of this procedure.
Thus, current treatment regimens for cirrhosis-related liver damage provide solutions for some of the complications accompanying cirrhosis while being useless in inducing liver repair and regeneration.
Liver Regeneration and Angiogenesis
Liver regeneration is a dynamic process in which proliferation of cells such as hepatocytes, biliary epithelial cells, and endothelial cells occurs. Liver tissue regeneration is believed to be controlled by various growth stimulating and inhibiting factors of autocrine or paracrine origin acting in concert; however, the exact role and mechanism of these factors is yet to be understood.
Liver tissue exhibits some self-regenerative properties, which are dependent on proper function of the complex vascular structure of the liver.
It has been observed that partial hepatectomy induces proliferation of all cells populations within the liver. Such cellular proliferation initiates in the periportal region (i.e. around the portal triads) and proceeds toward the centers of lobules. Proliferating hepatocytes initially form clumps, which soon transform into classical plates. Similarly, proliferating endothelial cells develop into the type of fenestrated cells typical of the sinusoids, which carry blood within the liver.
Liver angiogenesis, which is the formation of new blood vessels in liver tissue, is controlled by cells which secrete angiogenic and angiostatic factors (Yamane et al, 1994; Monacci et al, 1993).
One such angiogenic factor is vascular endothelial growth factor (VEGF) which posses endothelial-specific mitogenic and angiogenic properties and as such plays a multi-factorial role in development and maintenance of the vascular structure. In the liver, VEGF, which is secreted in a soluble form from hepatocytes, binds the VEGF-specific endothelial receptors, flt-1 and KDR/flk1 (Monacci et al, 1993; Jakeman et al, 1992). Although three VEGF receptors are known, the KDR/flk1 is probably the receptor functional in angiogenesis (Hanahan et al, 1997).
Little is known about the molecular regulation of angiogenesis and the communication of hepatocytes and sinusoidal endothelial cells in the liver.
Following partial hepatectomy VEGF expression is increased with a maximum level at 72 hours which is followed by expression of its receptors on sinusoidal endothelial cells (maximum 72-168 hours) (Mochida et al, 1996). Plasma VEGF levels are increased in acute hepatitis and in patients recovering from fulminant hepatitis (Akiyoshi et al, 1998). In addition, VEGF increased the rate of DNA synthesis in rat liver tissue following partial hepatectomy.
Hepatocyte growth factor (HGF) is another growth stimulating factor, which participates in liver regeneration. This growth factor which contributes to maintenance and proliferation of primary liver cells is expressed in the sinusoidal endothelial cells (Noji et al, 1990). It has been observed that HGF levels increase in the plasma of patients with hepatic failure and in the plasma or serum of animals with experimentally induced liver damage. The kinetics of this response is usually rapid, and precedes the first round of DNA synthesis during liver regeneration.
While reducing the present invention to practice, the present inventors have uncovered that localized over-expression of VEGF and HGF in liver tissue can be utilized to enhance liver regeneration, capillary production, and formation of sinusoidal network.
Thus, over-expression of such growth factors in liver tissue can be used to induce liver tissue regeneration and to prevent or alleviate portal hypertension thus providing an alternative treatment strategy for a variety of untreatable liver diseases.